Phenylacetyl-CoA ligase from penicillium chrysogenum

ABSTRACT

A process for preparing an enzyme from  Penicillium chrysogenum  having Phenylacetate-Coenzyme A activity. DNA encoding for the enzyme is also provided and its use in the production of modified strains.

This application is a national stage application (35 U.S.C. 371) ofPCT/EP96/02799, filed Jun. 26, 1996 which claims priority to GreatBritain application Ser. No. 9513403.7, filed Jun. 30, 1995.

The present invention relates to an enzyme useful in the synthesis ofpenicillins from intermediates involved in penicillin biosynthesis. Thepresent invention also relates to processes for the preparation of theenzyme and DNA coding for the enzyme.

The biochemical pathway for Penicillin G is disclosed in the literature(Queener (1990) Antimicrobial Agents and Chemotherapy 34(6), 943-948;Martin (1992) J. Industrial Microbiol 9, 73-90; Luengo (1995) J.Antibiotics 48 (11), 1195-1212). The pathway has been the subject ofconsiderable study with a view to increasing the yield (titre) infermentation processes.

Phenylacetate (PAA) and Phenoxyacetate (POA) are activated to thecorresponding CoA thioesters in Penicillium chrysogenum by a ligaseenzyme (e.g. PAA-CoA ligase). These thioesters are then used for thebiosynthesis of Penicillin G in the case of PAA and penicillin V in thecase of POA. PAA-CoA ligase is therefore thought to be essential in thebiosynthesis of these commercially important therapeutic antibiotics.

An enzyme from Pseudomonas putida having PAA-CoA ligase activity hasbeen isolated (J. Biol. Chem. 267(12), 7084-7090 (1990) in apurification procedure involving ammonium sulphate precipitation andpotassium chloride elution from a DEAE-Sephacel column. The enzyme has amolecular weight of 48 kDa +/−1 kD, a pH optimum of 8.2 and is involvedin PAA catabolism.

Attempts to assay an enzyme having PAA-CoA ligase activity from P.chrysogenum by the hydroxymate method (a colorimetric assay detectingphenylacetyl-hydroxamate or phenoxyacetyl-hydroxamate at 540 nm) havebeen reported by Kogekar & Deshpande (1983) Ind. J. Biochem. Biophys 20,208-212 and by Brunner & Rohr (1975) Methods Enzymol 43, 476-481;however other workers (Martinez-Blanco et al (1992) J. Biol. Chem.26(8), 5474-5481) are of the view that the protein had not been purifiedor the activity characterised in detail. Moreover the latter authorsfailed to find the enzyme by the reported procedure.

WO 96/10085 (Gist Brocades, published Apr. 4, 1996) reviews these andother attempts at isolating a PAA CoA ligase which operates in thepenicillin pathway. In WO 96/10085 an acyl-CoA enzyme synthetase isdescribed as being obtained from a strain of Penicillium chrysogenum B10 which is held by PanLabs (USA). Among the specific propertiesattributed to the enzyme are the following: molecular weight about 50kDa (as determined by gel filtration), pH optimum pH 8 to 8.5 (lowactivity at pH 7 or below), temperature optimum at 40° C., pI higherthan 7.25. Importantly the enzyme can be purified by ammonium sulphateprecipitation. It has a fairly wide specificity (ie. it is able tocatalyze the formation of phenoxyacetyl-coenzyme A,phenylacetyl-coenzyme A, adipyl-coenzyme A and hexanoyl-coenzyme A fromMg 2+, ATP, CoASH, and phenoxyacetic acid, phenylacetic acid, adipicacid or hexanoic acid respectively but does not show any significantactivity towards acetic acid. Also it is said that the enzyme isstabilised by reducing agents. A high concentration of ammonium sulphateor glycerol also stabilises the enzyme. No indications of purity aregiven for the enzyme activity obtained by the methods disclosed and nosequence or N-terminal sequence is given for the enzyme and thecorresponding DNA is not characterised or its sequence given.

In spite of all these efforts little is known about the authenticPAA-CoA ligase enzyme that operates in Penicillium sp. in vivo . It hadbeen speculated that the enzyme responsible may be involved in primarymetabolism (Smith et al (1990) Biotechnology 8, 39-41). Martinez-Blancoet al (1992) ibid selected one possible candidate enzyme, acetyl CoAsynthetase, and purified it from P. chrysogenum on the basis of acetylCoA synthetase activity. They were able to show that in addition toforming the CoA derivative of acetate, acetyl CoA synthetase was able toactivate several fatty acids (C2 -C8) and some aromatic molecules(including PAA) in vitro.

The acetyl CoA synthetase gene has been sequenced (International PatentWO92/07079, Gouka et al (1993) Appl. Microbiol. Biotechnol. 3, 514-519,Martinez-Blanco et al (1993) Gene 130, 265-270) and has been shown tohave homology with other acetyl CoA synthetases from fungi. Howevermutations in this gene selected by fluoroacetic acid do not appear toalter penicillin production levels (International Patent WO92/07079)suggesting that in vivo another enzyme is actually responsible foractivation of PAA.

In the present invention a direct assay for PAA-CoA ligase activity hasbeen developed in conjunction with a specific purification protocol andthis has allowed purification of and subsequent cloning of what isbelieved to be the authentic PAA-CoA ligase. The enzyme isolated by thepresent invention has a different N-terminal amino acid sequence to theacetyl CoA synthetase mentioned above and possesses a number ofdifferent properties (e.g. molecular weight) indicating that a differentenzyme has been isolated from all the enzymes attributed with this roleto date. The properties characteristic of the enzyme isolated in thepresent invention include an absolute dependence on CoASH as substratewhile the enzyme isolated by Kogekar & Deshpande (1983) ibid was assayedin conditions where CoASH was omitted. This and other differencesbetween the isolated proteins (e.g. pH optima and other characteristicsgiven in the examples below) show that the enzyme in the presentinvention is different to any of those described in the prior art. It isbelieved that the present work represents the first isolation of a pureform of the enzyme PAA CoA ligase from Penicillium sp. In particular thepresence of an SKI C-terminal peptide is consistent with this enzymehaving a real role in penicillin biosynthesis. The enzyme of the presentinvention differs from that in WO 96/10085 mentioned above in that ithas a different molecular weight and the enzyme of the presentinvention, unlike that of WO 96/10085 is sensitive to ammonium sulphateprecipitation and chloride salts.

Accordingly, the present invention provides an enzyme having PAA-CoAligase activity obtainable from Penicillium chrysogenum by culturing,harvesting and sonicating the mycelium, removing cell debris andfractionating the sonicate by anion-exchange chromatography, followed byhydrophobic interaction chromatography, affinity chromatography withsubstrate elution and gel filtration chromatography wherein the activechromatographic fractions are detected using a PAA and coenzyme Adependent assay.

The enzyme is preferably in purified form, advantageously insubstantially pure form.

The enzyme of this invention has an apparent molecular mass of 63 kDa(by SDS PAGE)

Preferably the enzyme includes the sequence of N-terminal amino acids

VFLPPKESGQLDP

In particular, the enzyme comprises the sequence of amino acids in FIG.1/ID SEQ 1

In a further aspect of the invention there is provided a method ofpreparing an enzyme having PAA-CoA ligase activity by culturingPenicillium sp. followed by extraction and purification wherein theactive fractions are detected using a PAA and Co-enzyme A dependentassay. In particular the Penicillium sp. is P. chrysogenum and themycelium is treated by sonication, followed by fractionation byanion-exchange, hydrophobic interaction, affinity and gel filtrationchromatography so as to provide an approximate 1000 fold increase inpurity.

The enzyme can be used in in vitro biotransformations. For example forCoA ester synthesis or for penicillin synthesis when mixed withacyl-CoA: 6-APA acyltransferase. The in vitro biotransformations can becarried out using whole cells, cell free extracts, permeabilised cellsor the isolated enzyme from the microorganisms or any of these inimmobilised form.

Where the biotransformation is carried out using whole cells, themicroorganism may be in the form of a growing culture, resting culture,washed mycelium, immobilised cells or protoplasts.

When cell-free extracts are used these are suitably produced by shearand /or chemical or enzymic lysis or other methods of disruption,preferably sonication, and optionally thereafter removing cell debris,leaving the enzyme activity in solution.

The enzyme is suitably prepared according to the examples below usingcommercially available strains of P. chrysogenum including wild typeNRRL 1 95 1. Other suitable strains of P. chrysogenum include highpenicillin producing strains e.g. strain BW1901 (EMBO J. 9(3), 741-747(1990) D. J. Smith et al.).

The enzyme may be prepared by culturing the microorganism in aconventional manner, especially under aerobic conditions in a suitableliquid or semi-solid medium. The culture conditions may be a temperaturein the range from 5-50° C. preferably 25-30° C. and pH in the range 3 to9, preferably 6-8, most preferably 7.2.

The enzyme may be isolated and used in purified form, partially purifiedform, as obtained in an impure state, as a filtrate from a disruptedcell preparation, as a crude cell homogenate and so on. Most suitablythe enzyme is, for example, at least purified to remove other enzymeswhich might also catalyse the destruction of the starting materials orthe enzyme.

Most suitably the enzyme is immobilised for example to an insolublesupport material such as by the procedures discussed by Powell (1990) inMicrobial Enzymes and Biotechnology ed. Fogarty & Kelly p369-394. Thisprovides the advantage of increased yield and throughput.

When the biotransformation is carried out using whole cells, a suitableincubation medium comprises medium: KH₂PO4 2 g, K₂HPO₄ 1.5 g, KCl 0.2 g,Mg Cl₂.6H₂O 0.2 g, Na₂SO₄.10H₂O 0.22 g, glucose 1.0 g in a liter ofdeionised water pH 6.5 or a water system with pH adjustment.

When the biotransformation is carried out using cell free extracts theincubation medium comprises a suitable buffer. In addition to substratesthe enzyme reaction mixture may contain one or more other cofactors e.g.metal ions or stabilisers. for example thiols.

The biotransformation may suitably be carried out in aqueous media, thereaction mixture suitably being maintained in the range pH 4-10 , moresuitably from 6 to 10, preferably around 9.0. The pH is suitablycontrolled using buffers or preferably by the addition of acid or basetitrant. The temperature of the reaction should generally be in therange 5-50° C. preferably 22-45° C., most preferably 30-37° C.Alternatively the reaction can be carried out in organic solvents or inthe presence of organic solvents e.g. acetone, methyl isobutyl ketone(MIBK).

The reaction time depends on such factors as concentrations of reactantsand cofactors, temperature and pH. After the reaction is complete theproduct can be isolated by conventional methods. The initialpurification conveniently involves a chromatography step.

In a further aspect the present invention also provides DNA encoding thePAA-CoA ligase of the present invention. The gene encoding said proteinis located within the DNA fragment shown in FIG. 2. In particular theDNA comprises substantially the DNA sequence in FIG. 3/ID SEQ 2.

In FIG. 2 the approximate length in kilobases (kb) of the DNA asdetermined by sizing experiments carried out by agarose gelelectrophoresis, is indicated. It should be understood that the figureis not intended to show all the restriction sites present on the DNA.

It will be understood that the DNA of this invention is not in itsnatural state as it occurs in nature but is in isolated or substantiallypure form. It will be understood that the invention encompasses DNAwhich may not have the precise configuration of restriction sitesillustrated if the said DNA has been derived by standard techniquesincluding nucleotide deletion, substitution, addition or inversion fromthe DNA according to any aspect of the invention described above.

Preferably the DNA of the present invention is derived from P.chrysogenum. However the invention also encompasses DNA sequencesderived from other suitable organisms especially producing organismsother than P. chrysogenum which sequences do not have the configurationof restriction sites shown but which hybridise, preferably underconditions of high stringency, with the DNA shown in FIG. 2 or asubfragment thereof and which code for PAA-CoA ligase or an enzyme withPAA-CoA ligase activity (high stringency conditions are for example, asgiven in Example 18).

The invention also provides a vector comprising such DNA, preferably anexpression vector for expressing PAA-CoA ligase in a suitable hostorganism. A specific example of such an expression vector is pBK-CMV(purchased from Stratagene) and used in this invention for expression inE. coli. In this invention the cDNA insert of PAA-CoA ligase (=pPEN09,FIG. 2) is a preferred vector for expression in E. coli.

The DNA of the invention and vectors containing same may find use inmany areas of industrial activity. That also applies to hostmicro-organisms transformed with said vectors and the enzymes theyexpress. For example the DNA may be utilised as a hybridization probe toidentify and isolate related or overlapping genes present on the totalcellular DNA of P. chrysogenum (NRRL 195 1) and of other micro-organismswhich produce enzymes of similar structure and specificity.

Recombinant vectors containing said DNA may be of value, whentransformed into suitable hosts, in the production of geneticallymodified micro-organisms which synthesize increased amounts ofpenicillin.

It would be very advantageous to increase the amount of activity ofPAA-CoA ligase in a suitable organism. Recombinant vectors could also beused in the generation of novel or hybrid antibiotics via the process ofgene transfer (see for example D. A. Hopwood et al, Nature, 1985, 314,642-644). Enzymes encoded by the DNA of the invention may be used, forexample, in cell-free systems especially when immobilised on suitablesolid supports, to prepare the known antibiotic from natural precursorsor a novel antibiotic from ‘unnatural’ precursors obtained, for example,by chemical synthesis.

The DNA of the invention or a fragment thereof (not necessarily carryingan intact gene) may be combined, either by recombinant DNA techniques orby natural recombination processes, with a fragment of a gene involvedin biosynthesis to produce a hybrid gene capable of directing thesynthesis of a hybrid enzyme. Such enzymes may be used in the productionof novel antibiotics by processes analogous to those hereinbeforedescribed.

The DNA of the invention may also be modified by the known techniques ofsite-directed mutagenesis (in a manner analogous to that described, forexample, by G. Winter et al, Nature, 1982, 299, 756-758; or by Zollerand Smith, Nucleic Acids Research, 1982, 10, 6487-6500) to give DNA inwhich specific mutations and/or deletions have been effected. Themutated DNA may be used to obtain an increased yield (or titre) ofpenicillin from a suitable host micro-organism.

The mutated DNA may also be used to obtain novel or hybrid antibioticsby gene transfer, or used in the production of mutant enzymes (muteins)which may be used in the production of novel antibiotics by analogousprocesses to those hereinabove described. The mutated DNA may also beused to alter other fermentation properties of suitable organisms e.g.PAA tolerance, altered substrates.

The following examples illustrate the invention.

EXAMPLE 1

P. chrysogenum Fermentation

Spores of Penicillium chrysogenum (SmithKline Beecham Strain BW1901) wasinoculated into 15 ml of PVS media (35 g/l corn steep liquor, 15 g/lglucose, 5 g/l CaCO₃, 8 ml/1 rape seed oil, pH to 5.9 with NaOH) in 100ml shake flask. The culture was grown for 48 h at 26° C. with orbitalshaking (230 rpm) before taking 1 ml of whole broth and transferring to10 ml of C5 media (35 g/l corn steep liquor, 85 g/l lactose, 10 g/lCaCO₃, 10 g/l NaH₂PO₄, 8 g/l(NH₄)₂SO₄, 4 g/l MgSO₄.7H₂O, 4 g/l Na₂SO₄, 6ml/1 rape seed oil, 6 g/l phenoxyacetic acid, pH to 6.0 with NaOH) in100 ml shakeflask. This culture was then grown for 55 h at 26° C. withorbital shaking (230rpm) before harvesting the mycelia.

EXAMPLE 2

Preparation of Protein Extracts from P. chrysogenum for Assay,Purification and Western Blotting of PAA-CoA Ligase.

Mycelia from a 55 h C5 shakeflask cultured as described in example 1 washarvested by filtering through glass microfibre filters (Whatman GFIA).The mycelial mat was washed with 300 ml of 0.9% (w/v) sodium chloride(4° C.) and then scraped from the filter and placed into 10 ml Ligaseassay buffer (30 mM Tris-HCl pH 9.0, 1 mM dithiothreitol, 100 μg/mlPefabloc™ in 50% glycerol). The mycelia was then sonicated on ice (3×15s burst using an Ultrasonics model W-385 sonicator, power setting 5,cycle rate 5 s, 50% duty cycle), and then the mycelial debris waspelleted by centrifugation (18000xg, 4° C. 30 min). The supernatant wasfrozen at minus 70° C. for storage or used immediately for PAA-CoALigase assay, purification or Western blotting (example 7).

EXAMPLE 3

In-Vitro Assay for PAA-CoA Ligase.

To demonstrate the presence of PAA-CoA Ligase activity in extracts orcolumn fractions, 20 μl were mixed with 0.1M phenylacetic acid in 50 mMTris-HCl pH 7.5 (20 μl), 0.1M sodium ATP (10 μl), 0.2M magnesiumchloride (10 μl), 0.02M sodium coenzyme A (10 μ) and 0.015Mdithiothreitol (10μl) in plastic eppendorf tubes. The tubes were vortexmixed (5 s) and then placed in a water bath at 30° C. for 15 min.Methanol (100 μl) was then added to the mixtures and the tubes werecentrifuged (14K, 1 min) to precipitate the proteins. The supernatantfractions were analysed for the presence of PAA-CoA by HPLC (example 4).In each set of assays a protein extract prepared from a P. chrysogenumshakeflask fermentation (example 1) was assayed as a positive controlalong with a PAA-CoA standard.

EXAMPLE 4

HPLC Analysis of Assay Supernatants.

Supernatant samples from the PAA-CoA Ligase assays (example 3) wereanalysed for the presence of PAA-COA using a Waters LCMI HPLC system.Samples (100 μl) were injected onto a Radial Pak C18 compression columnat room temperature with a flow rate of 2.5 ml/min using a mobile phaseof 0.2M sodium phosphate pH 5.4 (Buffer A) isocratically from 0-4 min.This was followed by a linear gradient to 100 % buffer B containing0.16M sodium phosphate pH 5.4 in 40 % acetonitrile (4-10 min). Buffer Bwas maintained at 100% for a further 2 min and then the system wasre-equilibrated ready for the next injection using a linear gradientback to 100% A (12-13 min). Peaks were detected at 260 nm and PAA-CoAhad a retention time of 12 min. Positive samples were those that hadco-eluting peaks with the PAA-CoA standard and showed the same UVabsorbance spectra as the standard as determined by a photodiode arrayspectrometer.

EXAMPLE 5

Purification of PAA-CoA Ligase

Care was required as the enzyme activity was found to be extremelylabile. Attempts at precipitation of the enzyme with ammonium sulphatewere unsuccessful as no activity could be found in the materialobtained. This in itself distinguishes the present enzyme from thatdescribed in WO 96/10085 (Gist-Brocades).

Unless otherwise stated the following procedure was conducted at 4° C.using buffer C containing 30 mM Tris-HCl, 4 mM DTT, 4 mM EDTA, 5 mMMgCl₂ and 20% glycerol (v/v) at pH 9.0. Separations were achieved usinga Pharmacia Hi-Load™ system. Cell free extract (500 ml), made as givenin example 1, was thawed slowly at 4° C. The extract was then adjustedto pH 9.0 using 5M NaOH with stirring on ice. To this stirring extractwas added 275 g of Q-Sepharose Fast Flow (Pharmacia) ion exchange mediawhich had been previously washed with 31 of water followed by 11 ofbuffer C. This mixture was stirred for 1.5 h on ice. The slurry was thenfiltered through a further 50 g of washed Q-Sepharose resin on a glasssinter using reduced pressure. The resin was then washed with a further100 ml of buffer C and then allowed to run dry giving 600 ml of clearextract containing PAA-CoA ligase. Ammonium sulphate (92.4 g ie.subprecipitation levels) was then added and the solution was stirred onice for 1 h followed by filtration (0.45 μm filter, Millipore, type HA).

The PAA-CoA Ligase extract (700m1) was then loaded at 1 ml/min onto aPhenyl-sepharose 6 Fast Flow, low substitution column (Pharmacia, 12cm×2.6 cm diameter) previously conditioned with 11 of buffer D (as forbuffer C with 140 g/l of ammonium sulphate). The loaded column was thenwashed with 230 ml of buffer D at 0.8ml/min and then eluted with alinear gradient from 100% buffer D to 100% buffer C over 238 ml at 0.8ml/min. 5.5 ml fractions were collected and tested for PAA-CoA ligaseactivity as given in example 3. Active fractions, 41 to 49 were pooledgiving 50 ml, which was loaded at 0.5 ml/min onto a 5 ml HiTrap™ Blueaffinity column (Pharmacia) previously washed with 50 ml of buffer C.The loaded column was washed with 15 ml of buffer C and then elutedusing a linear gradient from 100% buffer C to 100% buffer E over 25 mlat 0.5 ml/min. Buffer E was as for buffer C with the addition ofphenylacetic acid to given a final concentration of 0.5M which wasre-adjusted to pH 9.0 using solid NaOH. Elution from the affinity columnusing the natural substrate was used to increase purificationselectivity and to enable the enzyme activity to be measured in theresulting fractions. NaCl elution proved unsuccessful because this saltinhibited enzyme activity. In contrast the enzyme described in WO96/10085 appears to be stable when eluted in KCl.

Active fractions 21, 22 and 23 were pooled to give 6ml, which was thenseparated at 0.25 ml/min on a Sephacryl S-200 High Performance sizeexclusion column (Pharmacia, 64 cm×2.6 cm diameter) which had beenpreviously equilibrated in Buffer F (as for buffer C with adjustment topH 7.5 with 5M HCl). Active fractions 54 to 65 were pooled to give 11 mlwhich was concentrated down to 60 μl by centrifugal ultrafiltration(Centricon 10,000 MW cut-off, Amicon Inc.). Analysis of the fractionsfrom the size exclusion column by SDS-polyacrylamide gel electrophoresis(example 6) showed a protein of 63 kDa, the intensity of whichcorrelated with PAA-CoA ligase activity. Western Blotting (example 6)was used to complete the purification and to enable N-terminal aminoacid sequencing of the PAA-CoA Ligase.

EXAMPLE 6

Western Blotting of PAA-CoA Ligase Protein.

To provide material for N-terminal amino acid sequencing the purifiedprotein (60 μl, example 5) was mixed with an equal volume of SDS-PAGEsample buffer containing 0.5M Tris-HCl pH6.8 (10 ml), sodium dodecylsulphate (2 g, ultrapure), 2-mercaptoethanol (1 ml), glycerol (10 ml),distilled deionised water (7ml) and 0. 1% bromochlorophenol blue (2 ml).This mixture was boiled for 10 min and allowed to cool to roomtemperature. Aliquots (5, 15 and 20 μl) were loaded on to a 10%polyacrylamide gel (4% stacking gel) cast into a Bio-Rad Mini Protean IIelectrophoresis cell prepared using the Manufacturers protocol.Electrophoresis was conducted in electrode buffer containing 0.025MTris, 0.192M glycine and 0.1% w/v sodium dodecyl sulphate (ultrapure) at200V for 45 min after which time the polyacrylamide gel was placed in toelectroblotting buffer (10 mM 3-[cyclohexylamino]-1-propanesulfonicacid, pH11 in 10% methanol) for 5 min. Proteins in the polyacrylamidegel were transferred on to Applied Biosystems ProBlott™ immobilisationmembrane using a Bio-Rad Mini Trans-Blot electrophoretic transfer cellfollowing the Applied Biosystems protocol. On completion of blotting themembrane was stained with Coomassie Blue R-250 using the staining methodin the Applied Biosystems protocol. The protein band at 63 kDa was cutfrom the membrane and this material was used for N-terminal amino acidsequencing (example 7).

EXAMPLE 7

N-terminal Amino Acid Sequencing

N-terminal amino acid sequence was obtained from blotted protein(example 6) using an Applied Biosystems (ABI) 477A Pulsed LiquidSequencer. Sequence was obtained using standard Edman Chemistry withidentification of released PTH-labelled amino acids using ABI 120 Ånarrowbore chromatography. Analysis of the purified PAA-CoA ligaseprotein resulted in the following sequence assignment:

V-F-L-P-P-K-E-S-G-Q-L-D-P

EXAMPLE 8

Synthesis of PAA-CoA Ligase N-Terminal Peptide.

The N-terminal amino acid sequence determined from the 63 kDa PAA-CoALigase protein (example 7) was used to synthesise a peptide. This wassynthesized by Peptide and Protein Research Consultants (WashingtonSinger Laboratories, University of Exeter, Perry Road, Exeter, Devon EX44QG, UK) as 50 mg free peptides. 12 mg was conjugated to maleimideactivated BSA (Bovine Serum Albumin) using SMCC (Succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and 2.5 mg wasconjugated to maleimide activated OVA (Ovalbumin) using MBS(m-Maleimidobenzoyl-N-hydroxysuccinimide ester).

EXAMPLE 9

Production of Polyclonal Antibodies to Peptide Derived from theN-Terminus of the 63 kDa PAA-CoA Ligase Protein.

Peptide conjugated to BSA (example 8) was used for the production ofrabbit polyclonal antibodies specific to the 63 kDa PAA-CoA Ligaseprotein. The peptide-BSA conjugate (375 μg/ml) was filter sterilised(0.2 μm filter) and 1 ml of the sterile solution was thoroughly mixedwith 2 ml of non-ulcerative Freunds complete adjuvant (Brian MorrisInternational, Guildford U.K). A total of 0.8 ml of this mixture (100 μgof the peptide-BSA conjugate) was administered sub-cutaneously to NewZealand white rabbits (approximately 10 weeks old) at four differentinjection sites. Further immunisations were administered at 28 and 58days after the initial immunisation as described above with theexception that non-ulcerative Freunds incomplete adjuvant was used. Testbleed samples were taken from the marginal ear vein at 42 and 72 daysafter the initial immunisation to assess the antibody titre andspecificity using an Enzyme Linked Immunosorbent Assay (example 10).

EXAMPLE 10

Enzyme Linked Immunosorbent Assay (ELISA) for Determination of AntibodyTitre and Specificity to the 63 kDa PAA-CoA Ligase.

Determination of Antibody Titre

A 96 well flat bottomed microtitre plate (Nunc MaxiSorp™) was coatedwith PAA-CoA Ligase peptide-OVA conjugate (2001 μl/well, 0-10 μg/ml) inphosphate buffered saline pH 7.2 (8 g/l sodium chloride, 0.2 g/lpotassium chloride, 1.44 g/l sodium dihydrogen orthophosphate, 0.24 g/1potassium dihydrogen orthophosphate, pH 7.2). The coated microtitreplate was incubated at 4° C. for approximately 18 h after which time theplate was washed four times with wash buffer (10 mM Tris. 0.15M sodiumchloride, 0.02% sodium azide, 0.05% Tween 20 pH 7.2) using a DynatechMRW plate washer. This washing method was used throughout the rest ofthe procedure unless otherwise stated. Blocking buffer (1.56 g/l sodiumdihydrogen orthophosphate. 8.8 g/l sodium chloride, 0.2 g/l ficoll 400,0.2 g/l polyvinyl-pyrrolidone, 0.5% bovine gamma globulin's from Sigma,pH 7.4, 200 μl/well) was added to the plate and incubated at 37° C. in aDynatech Varishaker Incubator for 1 h All subsequent incubations at 37°C. were performed using this method. The plate was washed four times andthen rabbit polyclonal antibody (100 μl/well, 0-1:500 000 dilution),diluted in assay buffer (50 mM Tris, 150 mM sodium chloride, 1 mMmagnesium chloride, 0.5% BSA, 0.25% bovine gamma globulin's, 0.02%sodium azide, pH 7.4), was added to the plate and incubated at 37° C.After washing the plate an anti-rabbit IgG biotinylated antibody fromAmersham (100 μl/well, 1:5000 dilution in assay buffer) was added to theplate and incubated at 37° C. The plate was washed and a streptavidinalkaline phosphatase conjugate from Amersham (100 μl/well, 1:2000dilution in assay buffer) was added to the plate and incubated at 37° C.After washing the plate p-nitrophenyl phosphate (Sigma, 1 mg/ml)dissolved in 0.1 M glycine buffer (7.51 g/l glycine. 203 mg/l magnesiumchloride, 136 mg/l zinc chloride, pH10.4) was added to the plate(100μl/well) and incubated at 37° C. for 30 min. Sodium hydroxide (2M,50 μl/well) was then added to the plate and the absorbance of each wellwas measured at 405 nm using an Anthos Labtec plate reader. Antibodytitres between 1:500000 and 1:1000000 were obtained.

Determination of Antibody Specificity.

To demonstrate that the rabbit polyclonal antibodies would react withthe unconjugated PAA-CoA Ligase peptide an ELISA was performed asdescribed above with a modification at the incubation stage which usedthe rabbit polyclonal antibodies. The antibodies were diluted 1:100000-1: 400 000 in assay buffer and 50 μl of diluted antibody was addedto wells containing 50 μl of unconjugated peptide (0-20 μg/ml) preparedin assay buffer containing 2-mercaptoethanol (0.01%, v/v). 50%inhibition of antibody reactivity with the OVA-peptide conjugate wasachieved at an unconjugated peptide concentration of approximately 2μg/ml.

EXAMPLE 11

Determination of Antibody Specificity on Western Blots to PAA-CoA Ligasein Extracts from Penicillium chrysogenum, E. coli and on purifiedprotein samples.

Extracts prepared from Penicillium chrysogenum shakeflask cultures(BW1901 and BW1900A—a strain derived from a random mutation programme),from E coli JM109 and samples of purified PAA-CoA ligase protein wereWestern blotted as described in example 6 except that the membrane wasnot stained with Coomassie blue. Blotted membranes were placed inblocking buffer (1.56 g/l sodium dihydrogen orthophosphate, 8.8 g/lsodium chloride, 0.2 g/l ficoll 400, 0.2 g/1 polyvinyl-pyrrolidone, 0.5%bovine gamma globulin's, pH 7.4) and incubated for approximately 18 h at4° C. The membrane was then washed four times with wash buffer (10 mMTris, 0.15M sodium chloride, 0.05% Tween 20, pH7.2) before incubation(room temperature, 60 min) with the rabbit polyclonal antibodiesgenerated as given in example 9 and previously diluted 1:100 in assaybuffer (50 mM Tris, 150 mM sodium chloride, 1 mM magnesium chloride,0.5% BSA, 0.25% bovine gamma globulin's, pH7.4 ). After a further fourwashes in wash buffer the membrane was incubated with a donkeyanti-rabbit IgG horse radish peroxidase conjugate (Bio-Rad, 1:2000 inassay buffer, 60 min, room temperature) followed by a further fourwashes in wash buffer. The membrane was then incubated with a peroxidasesubstrate (Bio-Rad Horseradish Peroxidase Conjugate Substrate Kit) for10 min at room temperature before stopping the reaction by washing theblot in five changes of distilled deionised water. Positive PAA-CoALigase protein samples were those with a band at approximately 63 kDawhich comigrated with the purified PAA-CoA Ligase band. No positive bandat 63 kDa was detected in the E. coli JM109 protein extract.

EXAMPLE 12

Construction of a Penicillium chrysogenum cDNA bank.

A cDNA bank of Penicillium chrysogenum (SmithKline Beecham StrainBW1901) was constructed by following the instruction manual fromStratagene's ZAP Express TM cDNA Synthesis Kit. RNA was isolated fromstrain BW1901 as follows. Strain BW1901 grown as given in example 1 for40 h before harvesting the mycelia by filtration through two WhatmanGF/A 9.0 cm glass microfibre filters. The mycelia was washed by 100 mlof DEPC (diethylpyrocarbanate) treated distilled water. The mycelia wasthen ground in liquid nitrogen using a DEPC treated mortor and pestle.The frozen powdered mycelia was transferred to a 50 ml centrifuge tubecontaining 10 ml of solution G (4M guanidinium thiocyanate, 50 mMTris.HCl pH 7.5, 25 mM EDTA). The powder was resuspended in Solution Gand left on ice for 15 min. The mycelial debris was pelleted at 17500×gat 4° C. for 20 min. The supernatant was collected into another 50 mlcentrifuge tube and the RNA was extracted as Chomczynski and Sacchi(1987) Analytical Biochemistry 162, 156-159. From the total RNA, polyA⁺RNA was isolated by using CP Laboratories Mini-Oligo (dT) cellulose spincolumn kit as per the manufacturer's instructions. The poly⁺ RNA wasanalysed by gel electrophoresis as in Sambrook et al (1989) MolecularCloning, A Laboratory Manual (second edition). Aliquots (5 μg) of polyA⁺RNA were taken and used to synthesise double stranded cDNA flanked byEcoRI restriction site at 5′ end and a Xhol restriction site at the 3′end of the cDNA, by following Stratagene's ZAP Express™ cDNA synthesisprotocol. The cDNA thus synthesised was then size fractionated bypassage through Stratgene's Sephacryl S-400 spin columns supplied withthe cDNA synthesis kit following the manufacturer's instructions. Thesize fractionated cDNA was ligated into the XhoI and EcoRI arms of λZAPExpress as in Stratagene's Instruction manual. The ligated λDNA was thenpackaged by using Stratagene's Gigapack II Gold Packaging kit as per theinstruction protocol. The resulting cDNA phage were then amplified byplating out, incubating for 8 hours. Over 250,000 independent cloneswere obtained and the phage eluted in 20 ml of SM medium (0.1M NaCl,0.01M MgSO₄, 0.05M Tris-HCl pH7.5, 0.01% gelatine) per Nunc plate.aliquoted and stored as Sambrook et al. (1989) ibid.

EXAMPLE 13

Immunoscreening of cDNA Bank

The λZAP Express cDNA bank from Example 12 was screened for clones thatexpressed the 63 kDa PAA-CoA ligase protein using antibodies made asgiven example 9. The cDNA bank was screened as in Sambrook et al. (1989)ibid. The λZAP Express bank was infected at appropriate dilutions intothe E. coli strain XL1 Blue MRF′. The infected bacteria were grown for 4h on Luria agarose containing 10 mM MgSO₄, 0.2% maltose and 5 mM IPTG(to induce expression of the cDNA insert) at 37° C. before overlayingwith nitrocellulose (Hybond-C super, Amersham) and incubating overnightat 37° C. The filters were then immunoscreened using the primary rabbitantibodies (example 9) at {fraction (1/1000)} dilutions, while thesecondary antibody, goat anti-rabbit IgG alkaline phosphatase conjugateantibody (Sigma product A-8025) was used at {fraction (1/2000)}dilutions. The localisation of positive clones was performed withBCIP/NBT tablets (5 bromo4-chloro-3-indolylphosphate/nitrobluetetrazolium) purchased from Sigma (product B-5655) and used according tothe manufacturer's instructions. Twenty four positive clones wereidentified and cored, resuspended in SM buffer (5.8 g/l NaCl, 2 g/lMgSO₄.7H₂O, 50 mM Tris-HCl pH7.5, 0.01% gelatine) and rescreened at alower density of plaques until a positive signal could be identified toa single plaque.

EXAMPLE 14

Subcloning of Positive Clones

The positive λZAP Express cDNA clones identified from the immunoscreen(example 13) were then excised as plasmid pBKCMV derivatives using theExAssist helper phage and excision protocol provided by Stratagene. Theplasmid clones were then be grown up (kanamycin selection) and“mini-prepped” as given in Sambrook et al. (1989) ibid. The plasmidsobtained were then analysed by XbaI/SstI double digests in order tocharacterise the cDNA insert size of the clones. The cDNA insert sizeranged from 700 bp to 2.8 kb, with the largest group (12 clones)possessing an insert size of 2.0 kb. The plasmids were also digestedwith Sau3A (4 base pair cutter) to confirm grouping of clones on thebasis of similar restriction patterns. In this way the clones weredivided into 6 groups based on common restriction digest patterns.Representative clones from each group were then tested for production ofthe 63 kDa PAA-CoA ligase protein by Western analysis and also forenzyme activity.

EXAMPLE 15

Demonstration of Positive cDNA Clones Using Western Blotting.

Protein extracts from representative excised immunoscreen positiveclones were prepared by taking an overnight culture of the E. coilclones grown in Luria broth plus kanamycin (504 g/ml), IPTG (5mM) at 25°C. and adding an equal volume of SDS-PAGE sample buffer followed byboiling and electrophoresis as in example 6. The proteins were thenWestern blotted to determine the presence of the 63 kDa PAA-CoA ligaseprotein (example 11). Immunostaining of the membrane was performed asdescribed in example 11 other than that an anti-rabbit IgG alkalinephosphatase conjugate was used (1:2000 dilution in assay buffer) and thephosphatase substrate was BCIP/NBT supplied in tablet form (Sigma) andused according to the manufacturers instructions. One group of clones(cDNA insert size of 2.0 kb) was identified as having a 63 kDa protein(co-migrated with BW1901 control).

EXAMPLE 16

Assay of E. coli Clones for Ligase Activity.

Broth containing E. coli cells (grown as in example 15) were centrifugedin a refrigerated Denley centrifuge at 4000×g, 4° C. for 7 min to pelletthe cells. The supernatant was discarded and the cells resuspended inligase assay buffer (example 2) at half of the original broth volume.The cells were then chilled on ice before sonicating to disrupt thecells. Conditions for sonication were output 5, 50% duty cycle on anApollo Electronics Sonicator for 30 s bursts over 7 min on ice. Extractswere either used immediately or stored at −80° C. until used. PAA-CoAligase activity was demonstrated using the assay procedure described inexample 3 except that 40 μl of extract was used and the reaction mixturewas incubated for 60 min at 30° C. The presence of PAA-CoA in the assaysupernatants was demonstrated using HPLC as described in example 4 withthe addition of a Waters 996 photodiode array detector for spectralanalysis. PAA-CoA ligase activity was detected in clone 6.6(representative of the group with a cDNA insert size of 2.0 kb) and thePAA-CoA produced was confirmed by diode array analysis against a PAA-CoAstandard.

EXAMPLE 17

Sequence of 5′ DNA of PAA-CoA Ligase Clones.

Clone 6.6 (=pPEN09) was “maxi-prepped” and DNA purified by CsClgradients as in Sambrook et al. (1989) ibid. A restriction map of pPEN09was prepared by performing single and double enzyme digests (FIG. 2).This clone was then sequenced using the primer(5′-ACAGGAAACAGCTATGACCTTG-3′) purchased from Cruachem and usingPharmacia's T7 sequencing kit and following the manufacturer'sinstructions provided. The sequence of the 5′ end of the cDNA insert,shown below, verified that the translated amino acid sequence at theN-terminus of the cDNA matched that of the peptide sequence previouslyobtained (example 6). except for the starting methionine (absent fromthe peptide sequence).

TCTAAACCCCGAGATCACCTCAGTTTCCTGCACTTTGGAGACCTGCCC −26CTATATTACCCCGAGGATTTGGGAAA ATG GTT  TTT TTA CCT 15                           M   V    F   L   P 5 CCA AAG GAG TCC GGT CAATTG GAC CCA ATT CC 48 P   K   E   S   G   Q   L   D   P   I   P 16 GACAAT ATT CCA ATC AGC GAG TTT ATG CTC AAT 81D   N   I   P   I   S   E   F   M   L   N 27

The remainder of pPEN09 was sequenced using standard dideoxynucleotidetermination reactions containing 7-deaza dGTP. [³⁵S]dATP was used as thelabel. Sequence reactions were analysed on 6% polyacrylamide wedge gelscontaining 8M urea [Sanger et al (1977) PNAS 74, 5463-5467; Chen andSeeburg (1985) DNA 4, 165-170]. Nested deletions were generated fromboth the T7 and T3 ends using Exoll and SI nuclease [Henikoff (1984)Gene 24, 351-359]. The deletion clones were size selected for DNAsequencing by electrophoresis on agarose gels. The selected clones beingsequenced as pPEN09. Internal sequencing primers were synthesised asnecessary. The complete sequence of the cDNA insert is shown in FIG. 3.The translated protein sequence is shown in FIG. 1.

Comparison of the deduced amino acid sequence of the PAA-CoA ligaseprotein with the National Biomedical Research Foundation Proteinsequence database (NBRF-PIR) and SWISS-PROT Protein Sequence Data Bank(SWISS-PROT) on DNASTAR software gave the best match with4-coumarate-CoA ligase from potato. Using the DNASTAR megalign programme(clustal method) the PAA-CoA ligase protein and the potato4-coumarate-CoA ligase were aligned. Analysis of the sequence distancesrevealed a 25% similarity of amino acids between the two proteins. Asimilar comparison with acetyl CoA synthetases from fungi (Penicillium,Aspergillus, Neurospora and yeast) showed only a 15% similarity.

The last three amino acids of the PAA-CoA ligase protein areserine-lysine-isoleucine. This amino acid sequence fits the consensusfor the C-terminus Microbody Targeting Signal (CMTS) and the same aminoacids have been considered essential for targeting the Hansenulapolymorpha catalase protein to the microbodies (Didion & Roggenkamp(1992) FEBS Lett. M(2-3), 113-116). The SKI C-terminal tripeptide canalso target protein to the microbody in Neurospora crassa (de Zoysa &Connerton (1994) Curr. Genet. 26, 430437). The last step of penicillinbiosynthesis carried out by the ACTF protein (utilising PAA-CoA—theproduct of PAA-CoA ligase) has been localised to microbodies in P.chrysogenum (Muller et al. (1991) EMBO J. 10(2) 489-495). The ACTFprotein also has a CMTS that is required for targeting to the microbody(EP 0488 180 A2). That the PAA-CoA ligase protein has a CMTS isconsistent with its role in penicillin biosynthesis.

EXAMPLE 18

Hybridisation of Genomic DNA with PAA-CoA Ligase cDNA Probe

Genomic DNA from a number of strains, including Penicillium chrysogenumwild-type NRRL1951, BW1900A, BW1901 was isolated as follows. The strainswere grown as in example 1, and harvested after 40 h by filtrationthrough Whatman GF/A glass microfibre filters. The mycelia was rinsed in0.9M NaCl before freezing in liquid nitrogen. The mycelia was ground toa fine powder in liquid nitrogen and the powder resuspended in solutionG (example 12) leaving on ice for 15 min. The mycelial debris waspelleted at 17500×g at 4° C. for 20 min. The supernatant was collectedinto another 50 ml centrifuge tube and the DNA was extracted withphenol/chloroform pH8.0, extracted with chloroform and ethanolprecipitated as in Sambrook et al (1987) ibid. The nucleic acid wasresuspended in 10 mM Tris.HCl pH8.0, 1 mM EDTA and the RNA was removedby RNase treatment as in Sambrook et al (1989) ibid. The genomic DNAswere digested with BamHI and the digests were electrophored, blottedonto Hybond N membrane (Amersham) as in Sambrook et al (1989) ibid. Themembrane was hybridised with the cDNA insert from pPENO9 as follows: Themembrane was pre-hybridised in 6×SSC, 1% SDS, 6% PEG6000, 100 μg/mldenatured, fragmented Herring Sperm DNA at 60° C. for 6h. After thistime labelled, denatured cDNA fragment (using Amersham's Megaprime kitand ³²P-dCTP as manufacturer's instructions) was added to thehybridisation solution and the hybridisation continued at 60° C.overnight. The membranes were then washed at 65° C. in 2×SSC, 0.1% SDSfor 30 min (twice). Membranes were autoradiographed as Sambrook et al(1989) ibid. Results showed a single common 8 kb BamHI fragment from allthe Penicillium strains (including wild-type NRRL 1951) that hybridisedto the cDNA probe.

EXAMPLE 19

Construction of λEMBL3 Bank

A shakeflask culture of P. chrysogenum SmithKline Beecham strain BW1900Awas prepared by inoculating a loopful of spores into 50 ml of ACM media(20 g/l malt extract, 1 gal bacto-peptone, 20 g/l glucose) andincubating with shaking at 25° C. After 40 h growth the mycelia washarvested by filtration through Whatman GF/A glass microfibre filtersand rinsed in 0.9M NaCl. The mycelia was then resuspended in 0.9M NaClcontaining 10 mg/ml Novozym (Novo Biolabs, Novo Industri. Denmark) andincubated at 25° C. for 2 h. The protoplasts were purified from themycelial debris by passage through a cotton wool filter beforecentrifuging at 4000×g for 10 min to pellet the protoplasts. Theprotoplasts were rinsed twice in 0.9M NaCl before adding 4M guanidiniumthiocyanate, 500 mM Tris.HCl pH7.5, 25 mM EDTA to lyse the protoplasts.The debris was removed by centrifugation and the supernatant containingchromosomal DNA, was added to an equal volume of 8M LiCl mixed gentlyand stored at −20° C. for 30 min. The protein and some RNA was thenpelleted by centrifugation (1000×g, 4° C., 10 min) and the supernatantcontaining chromosomal DNA was ethanol precipitated. The chromosomal DNAwas partially digested with Sau3A and the fragmented chromosomal DNA wassize fractionated on a sucrose density gradient as in Sambrook et al(1989) ibid. Fractions containing Sau3A fragments greater than 10 kbwere pooled and used in the construction of a λEMBL3 bank. The Sau3Agenomic fragments were ligated to the λEMBL3 BamHI arms obtained fromPromega. The ligated %DNA was then packaged using Promega's Packagenekit. The packaged λDNA was then amplified by infecting E. coli strainLE392 cells and the plaques were plated out onto a bacterial lawn andincubating for 8 h at 37° C. as Sambrook et al (1989) ibid.Approximately 18000 independent clones were obtained. The phage wereeluted into SM buffer and stored appropriately (as in Sambrook et al.,1989. ibid).

EXAMPLE 20

Cloning the Genomic DNA of PAA-CoA Ligase

From the cDNA clone 6.6 a SstI-XbaI fragment containing the cDNA insert(FIG. 2) was used to probe a λEMBL3 bank (prepared in example 19) byplaque hybridisation as Sambrook et al (1989) ibid (conditions same asin example 18). From the primary screen a number of primary positiveswere identified and these were picked and used for a secondary screen atlower dilutions. The plaque hybridisation was repeated and individualplaques were identified as hybridising to the PAA-CoA ligase cDNA probe.These λEMBL clones were picked off and amplified. The λEMBL clones weredigested with one of BamHI, EcoRI or SaII and all pairwise combinations,along with single digests of genomic DNA from strain BW1900A. Thedigests were electrophoresed, blotted and characterised by Southernhybridisation and restriction fragments containing the whole PAA-CoAligase gene were identified. A 6.5 kb EcoRI fragment was taken from someof the λEMBL clones (same size fragments seen in chromosomal DNAdigests) and subloned into pIJ2925 (G. R. Janssen and M. J. Bibb (1993)Gene 124 (1) 133-134). A restriction map of the genomic DNA is presentedin FIG. 4.

EXAMPLE 21

Transformation of P. chrysogenum Strain BW1901 with PAA-CoA Ligase

The 6.5 kb EcoRI subclone in pIJ2925 (example 20, =pAMX131) was usedwith a linear amdS fragment from p3SR2 (Hynes et al (1983) Mol. Cell.Biol. 3, 1430-1439) to co-transform protoplasts of P. chrysogenum strainBW1901 (method as Tilburn et al. (1984) Gene 26, 205-221). Transformantswere selected for the ability to utilise acetamide. The transformantswere then screened for PenV titre. A number of transformants had greaterlevels of PenV compared to BW1901 control (up to 111% on retests),possibly suggesting the integration of both plasmids. Such a resultwould support the role of this clone in penicillin biosynthesis.

6 1 578 PRT Penicillium Chrysogenum 1 Met Val Phe Leu Pro Pro Lys GluSer Gly Gln Leu Asp Pro Ile Pro 1 5 10 15 Asp Asn Ile Pro Ile Ser GluPhe Met Leu Asn Glu Arg Tyr Gly Arg 20 25 30 Val Arg His Ala Ser Ser ArgAsp Pro Tyr Thr Cys Gly Ile Thr Gly 35 40 45 Lys Ser Tyr Ser Ser Lys GluVal Ala Asn Arg Val Asp Ser Leu Ala 50 55 60 Arg Ser Leu Ser Lys Glu PheGly Trp Ala Pro Asn Glu Gly Ser Glu 65 70 75 80 Trp Asp Lys Thr Leu AlaVal Phe Ala Leu Asn Thr Ile Asp Ser Leu 85 90 95 Pro Leu Phe Trp Ala ValHis Arg Leu Gly Gly Val Leu Thr Pro Ala 100 105 110 Asn Ala Ser Tyr SerAla Ala Glu Leu Thr His Gln Leu Leu Asp Ser 115 120 125 Lys Ala Lys AlaLeu Val Thr Cys Val Pro Leu Leu Ser Ile Ser Leu 130 135 140 Glu Ala AlaAla Lys Ala Gly Leu Pro Lys Asn Arg Ile Tyr Leu Leu 145 150 155 160 AspVal Pro Glu Gln Leu Leu Gly Gly Val Lys Pro Pro Ala Gly Tyr 165 170 175Lys Ser Val Ser Glu Leu Thr Gln Ala Gly Lys Ser Leu Pro Pro Val 180 185190 Asp Glu Leu Arg Trp Ser Ala Gly Glu Gly Ala Arg Arg Thr Ala Phe 195200 205 Val Cys Tyr Ser Ser Gly Thr Ser Gly Leu Pro Lys Gly Val Met Ile210 215 220 Ser His Arg Asn Val Ile Ala Asn Thr Leu Gln Ile Lys Ala PheGlu 225 230 235 240 Gln Asn Tyr Arg Asp Gly Gly Gly Thr Lys Pro Ala SerThr Glu Val 245 250 255 Ala Leu Gly Leu Leu Pro Gln Ser His Ile Tyr AlaLeu Val Val Ile 260 265 270 Gly His Ala Gly Ala Tyr Arg Gly Asp Gln ThrIle Val Leu Pro Lys 275 280 285 Phe Glu Leu Lys Ser Tyr Leu Asn Ala IleGln Gln Tyr Lys Ile Ser 290 295 300 Ala Leu Phe Leu Val Pro Pro Ile IleIle His Met Leu Gly Thr Gln 305 310 315 320 Asp Val Cys Ser Lys Tyr AspLeu Ser Ser Val Thr Ser Leu Phe Thr 325 330 335 Gly Ala Ala Pro Leu GlyMet Glu Thr Ala Ala Asp Phe Leu Lys Leu 340 345 350 Tyr Pro Asn Ile LeuIle Arg Gln Gly Tyr Gly Leu Thr Glu Thr Cys 355 360 365 Thr Val Val SerSer Thr His Pro His Asp Ile Trp Leu Gly Ser Ser 370 375 380 Gly Ala LeuLeu Pro Gly Val Glu Ala Arg Ile Val Thr Pro Glu Asn 385 390 395 400 LysGlu Ile Thr Thr Tyr Asp Ser Pro Gly Glu Leu Val Val Arg Ser 405 410 415Pro Ser Val Val Leu Gly Tyr Leu Asn Asn Glu Lys Ala Thr Ala Glu 420 425430 Thr Phe Val Asp Gly Trp Met Arg Thr Gly Asp Glu Ala Val Ile Arg 435440 445 Arg Ser Pro Lys Gly Ile Glu His Val Phe Ile Val Asp Arg Ile Lys450 455 460 Glu Leu Ile Lys Val Lys Gly Leu Gln Val Ala Pro Ala Glu LeuGlu 465 470 475 480 Ala His Ile Leu Ala His Pro Asp Val Ser Asp Cys AlaVal Ile Ala 485 490 495 Ile Pro Asp Asp Arg Ala Gly Glu Val Pro Lys AlaIle Val Val Lys 500 505 510 Ser Ala Ser Ala Gly Ser Asp Glu Ser Val SerGln Ala Leu Val Lys 515 520 525 Tyr Val Glu Asp His Lys Ala Arg His LysTrp Leu Lys Gly Gly Ile 530 535 540 Arg Phe Val Asp Ala Ile Pro Lys SerPro Ser Gly Lys Ile Leu Arg 545 550 555 560 Arg Leu Ile Arg Asp Gln GluLys Glu Ala Arg Arg Lys Ala Gly Ser 565 570 575 Lys Ile 2 1976 DNAPenicillium Chrysogenum 2 gaattcggca cgagtctaaa ccccgagatc acctcagtttcctgcacttt ggagacctgc 60 ccctatatta ccccgaggat ttgggaaaat ggtttttttacctccaaagg agtccggtca 120 attggaccca attcccgaca atattccaat cagcgagtttatgctcaatg agagatatgg 180 acgagtgcga cacgccagct cccgggaccc atacacctgtggtattaccg ggaagtcata 240 ctcgtcgaaa gaggtagcca atcgcgtcga ctcgctggctcgtagtctat caaaggaatt 300 tggttgggcg ccgaatgaag ggtcagaatg ggataagacattggccgtgt ttgccctcaa 360 cactatcgat tccttacccc tattctgggc cgttcacagactgggcggtg ttctcactcc 420 cgccaacgca tcatactccg ccgccgagct gacgcatcagctgcttgatt ccaaggccaa 480 ggcccttgtg acttgtgttc ctctcctctc catctcactggaagctgcag ccaaagctgg 540 tctcccgaag aacagaatct acttactcga tgtacctgagcagcttcttg gcggagtcaa 600 gcctccagca ggatacaagt ccgtttccga actgacccaggctgggaagt ctctcccgcc 660 agtggatgaa ttgcgatgga gcgcgggtga aggtgcccggcgaacagcat ttgtgtgcta 720 ctcaagtgga acgtctggat tgccgaaagg agtcatgatctcacaccgca acgtgatcgc 780 caataccctt cagatcaagg cgtttgagca gaactaccgggatggtgggg gcacaaagcc 840 tgcgagtact gaggttgctc ttggtctcct tccgcagagccatatctatg ctcttgtggt 900 cattggccat gctggggcat accgaggcga ccaaacaatcgttctcccca aattcgaatt 960 gaaatcctac ctgaacgcca tccaacagta caagatcagtgcgctgttcc tggtacctcc 1020 gatcatcatt cacatgctgg gcactcaaga cgtgtgctccaagtatgacc tgagttccgt 1080 gacgtctctg ttcacgggag cggcacccct gggtatggagacagctgccg atttcctcaa 1140 actctacccg aacattttga tccgccaagg atacggtctgacagagacat gcacggtcgt 1200 aagctcgacc cacccgcacg atatctggct aggttcatccggcgctttgc tccctggagt 1260 cgaggcacga attgtgacgc ctgaaaacaa ggaaatcacaacgtacgact caccgggcga 1320 attggtggtc cgaagcccaa gcgtcgtcct gggctatttgaacaacgaaa aagccaccgc 1380 agagacattt gtggacggat ggatgcgtac gggagacgaggctgtcatcc gtagaagccc 1440 gaagggcatc gagcacgtgt ttattgtcga tcggatcaaggagttgatca aggtcaaggg 1500 tctgcaagtc gcgcctgccg aactcgaagc ccatatcctcgcccaccccg atgtctcgga 1560 ctgtgctgtc atcgctattc cggatgatcg tgcaggagaagtacccaagg ccattgttgt 1620 gaagtccgcc agcgcaggat cggacgaatc tgtctcccaggctctcgtga agtatgttga 1680 ggaccacaag gctcgtcaca agtggttgaa gggaggtatcagatttgtgg atgccattcc 1740 caagagcccg agtggtaaga ttcttcgtcg gttgatccgtgaccaagaga aggaggcacg 1800 gagaaaggct ggtagcaaga tctaaaaatg tcgggggtagctttgattag aacttggtct 1860 gggaaacttg gaaaccgata accattgttg gcttgaactagaagtatata tgtaaatacg 1920 tgataaacaa ggcatctcat ctgctgttaa aaaaaaaaaaaaaaaaaaaa ctcgag 1976 3 13 PRT Penicillium Chrysogenum 3 Val Phe LeuPro Pro Lys Glu Ser Gly Gln Leu Asp Pro 1 5 10 4 22 DNA PenicilliumChrysogenum 4 acaggaaaca gctatgacct tg 22 5 155 DNA PenicilliumChrysogenum 5 tctaaacccc gagatcacct cagtttcctg cactttggag acctgcccctatattacccc 60 gaggatttgg gaaaatggtt tttttacctc caaaggagtc cggtcaattggacccaattc 120 ccgacaatat tccaatcagc gagtttatgc tcaat 155 6 27 PRTPenicillium Chrysogenum 6 Met Val Phe Leu Pro Pro Lys Glu Ser Gly GlnLeu Asp Pro Ile Pro 1 5 10 15 Asp Asn Ile Pro Ile Ser Glu Phe Met LeuAsn 20 25

What is claimed is:
 1. An isolated polypeptide having PAA-CoA ligaseactivity obtainable from Penicillium chrysogenum by culturing,harvesting and sonicating the mycelium, removing cell debris andfractionating the sonicate by anion-exchange chromatography, followed byhydrophobic interaction chromatography, affinity chromatography withsubstrate elution and gel filtration chromatography wherein the activechromatographic fractions are detected using a PAA and coenzymeA-dependent assay.
 2. A polypeptide according to claim 1 having anapparent molecular mass of approximately 63 kD by SDS PAGE.
 3. Apolypeptide according to claim 1 incorporating the sequence ofN-terminal amino acids: V-F-L-P-P-K-E-S-G-Q-L-D-P [SEQ ID NO:3].
 4. Apolypeptide according to claim 1 which comprises the sequence of aminoacids shown in SEQ ID NO:1.
 5. A process for preparing an enzymeaccording to claim 1 which comprises culturing Penicillium sp., followedby extraction and purification wherein the active fractions are detectedusing a PAA and Co-enzyme A dependent assay.
 6. An isolated polypeptidehaving PAA-CoA ligase activity obtainable from Penicillium chrysogenumincorporating the sequence of N-terminal amino acids:V-F-L-P-P-K-E-S-G-Q-L-D-P [SEQ ID NO:3].
 7. An isolated polypeptidehaving PAA-CoA ligase activity obtainable from Penicillium chrysogenumwhich comprises the sequence of amino acids shown in SEQ ID NO:1.
 8. Anisolated DNA encoding a polypeptide having PAA-CoA ligase activityobtainable from Penicillium chrysogenum by culturing, harvesting andsonicating the mycelium, removing cell debris and fractionating thesonicate by anion-exchange chromatography, followed by hydrophobicinteraction chromatography, affinity chromatography with substrateelution and gel filtration chromatography wherein the activechromatographic fractions are detected using a PAA and coenzymeA-dependent assay.
 9. The DNA according to claim 5 having theconfiguration of restriction sites as shown in FIG. 2 or
 4. 10. The DNAaccording to claim 9 which comprises the DNA sequence in SEQ ID NO:2.11. An isolated DNA, and fragments thereof, encoding a polypeptidehaving PAA-CoA ligase activity which hybridises under conditions of highstringency with the DNA according to claim 8 as follows: prehybridizedat 60° C. 6×SSC, hybridized overnight at 60° C. and washed at 65° C. in2×SSC.
 12. A vector comprising DNA according to any one of claims 8-11for expressing an enzyme having PAA-CoA ligase activity in a suitablehost organism.
 13. A host transformed with the vector of claim
 12. 14. Aprocess for producing penicillin comprising, culturing the transformedhost according to claim 13 and recovering penicillin from the culture.15. An isolated DNA encoding a polypeptide having PAA-CoA ligaseactivity obtainable from Penicillium chrysogenum which incorporates thesequence of N-terminal amino acids: V-F-L-P-P-K-E-S-G-Q-L-D-P [SEQ IDNO:3].
 16. An isolated DNA encoding a polypeptide having PAA-CoA ligaseactivity obtainable from Penicillium chrysogenum which comprises thesequence of amino acids shown in SEQ ID NO:1.